EP1203811A2 - Production of metabolites of interest by co-culture of plant cells and non-plant cells - Google Patents

Abstract

The invention relates to in vitro stable co-cultures of cells of plant origin and phythopathogens, for producing plant substances. The invention also relates to a method of co-culturing cells of plant origin and phytopathogens in a fermenter to produce substances of interest.

Description

The present invention relates to cell culture methods for the synthesis of new compounds, based on the co-culture of cells plants and cells or organisms from a different kingdom.

The superior plants and the mushrooms, by the richness of their genetic heritage, are capable of producing compounds capable to be of interest in many biotechnological applications, especially in the food, agrochemical, pharmaceutical or cosmetic. They are indeed a potentially inexhaustible source new molecules. When such a substance with activity interesting is identified, the question of its synthesis in sufficient quantity to meet existing needs should be considered.

Two main modes of production are possible: chemical synthesis, often low cost when it is relatively uncomplicated, and the extraction of the compound from plant or fungal biomass. In vitro culture is a technique used to synthesize these substances: by avoiding climatic vagaries and agronomic techniques, it makes it possible to produce in a uniform manner and sometimes with high yields. It also allows the activation of certain metabolic pathways suppressed during normal plant growth. However, in vitro culture also poses technological challenges such as axenic cultures in fermenters over long periods, with high generation times for plant cells.

Many molecules of interest, produced by plant cells and fungal in culture, are derivatives of primary metabolism. These derivatives are not directly necessary for the growth of the organism, have a wide variety of biological structures and activities, due to the diversification of the base units engaged in the synthesis routes. They are specific and vary from one species to another. Their synthesis is not done not throughout the life cycle but is linked to the state of the culture: it does not begins only when this state is reached, usually during the phase growth stationary. It is controlled by a set of genes regulating the timing and level of expression. Control mechanisms are integrated into the physiology of the producing organism.

The synthesis of the metabolites considered can induce more or less antagonism. less marked with the achievement of the primary metabolism corresponding to cell division. It generally takes place in two phases: a biomass production step then a step of manufacturing the metabolites in a special environment where the tissues are no longer growing.

The in vitro culture of plant cells and / or tissues is an important potential source of secondary metabolites, but their natural synthesis is often relatively weak: their expression is suppressed, and it is sometimes necessary to add specific inducers to the cells to express their dormant genetic heritage and optimize production.

Extracts of inactivated phytopathogens can thus play the role of elicitors and stimulate the production of interesting molecules. Elicitation consists in inducing an increase in the synthesis of certain secondary metabolites by plant cells; during an infection by an external pathogen, certain genes whose activity is weak, are stimulated, which leads to an increase in secondary metabolism and the synthesis of phytoalexins by the plant. These phytoalexins are anti-microbial substances produced and accumulated in response to aggression by the phytopathogenic organism. These are compounds derived from secondary metabolism and generally having a low molecular weight ( Paxton 1981, Whitehead and Threlfall 1992 ).

les éliciteurs biotiques : extraits naturels inactivés (par autoclavage ou congélation) comme des broyats de bactéries, de champignons phytopathogènes (hydrates de carbones dérivant de leur paroi cellulaire...)

les éliciteurs abiotiques : contraintes et stress dus au froid, aux radiations U.V ...

The addition of elicitors to plant cell cultures is therefore envisaged to improve the production of molecules of interest. There are two types of elicitor:

biotic elicitors: inactivated natural extracts (by autoclaving or freezing) such as ground bacteria, phytopathogenic fungi (carbohydrates derived from their cell walls, etc.)

abiotic elicitors: constraints and stresses due to cold, UV radiation ...

The elicitation therefore aims to make plant cells produce secondary metabolites of interest, in large quantities. In case there is already a natural expression of these metabolites, it's about trying to increase this by adding extracts of biotic elicitors in the feeding medium. Otherwise, it is to stress the strains so that they express their “dormant” genetic heritage in conditions comfort. Several methods are possible: adding precursors of the metabolites considered, their coupling with elicitors, the permeabilization or cell immobilization.

The table below presents examples of molecules of interest obtained by elicitation and the elicitor used.

Thus, under conditions of stress or external aggression, plants can activate certain metabolic pathways and reveal repressed genetic resources under normal growth conditions. For example, it is known that the production of certain quinones is effective only when the plant is attacked by bacteria or fungi. This is the case for most phytopathogens such as Verticillium dahliae (fungus) when it parasitizes Dahlia or the genus Aspergillus. Many bacterial taxa like the genus Erwinia produce the same effects. Attacks by insects (galls of oak) or viruses (red pigments on lime leaves) also cause defense reactions capable of generating new colored molecules. The plant reacts by secreting polyphenol oxidases (laccases and catecholases) which oxidize their polyphenols to quinones. The latter polymerize spontaneously on contact with oxygen in the air, creating a bacteriostatic and / or fungistatic "scar" film, thus avoiding the invasiveness of the exogenous aggressor.

In Cocoa, there is secretion of phytoalexins (arjunolic acid, cyclo-octa-sulfide, phenols). In Cotton, there is induction of HMGR (3-Hydro-3-Methyl-Glutaryl Co A Reductase), the first enzyme involved in the primary defense mechanism and synthesis of terpene. The plant also produces a sesquiterpenoid phytoalexin (isoprenoid) as a resistance factor, and deoxyhemigossypol in response to infection; we also observe a peroxidation of the lipid membrane, the decrease in the concentration of soluble proteins, a variation in the lipid content of the roots (decrease in the content of total lipids, neutral and phospholipids), and the synthesis of phenolic pigments (Yunosova et al. 1989, Li et al . 1995). On the eggplant, there is an increase in the activities of β-1,3-glucanase and amylase in the leaves. In potatoes, the response to infection results in hypersensitivity, the synthesis of a phytoalexin (rishidine), and suberization.

A similar phenomenon is observed in phytopathogens. When a fungus is in contact with a plant, its germ tube forms a specialized organ, the appressorium, which serves as a base for the penetration of the cuticle of plant cells. It is the combined action of mechanical pressure and various enzyme systems that allows this penetration. Thus, Verticillium dahliae produces an extracellular alkaline protease when it is growing in a liquid medium supplemented with a protein source. It also has pecto- and proteolytic enzymes: endo-poly-galacturonase, pectin trans-eliminase, pectin methyl-esterase playing a role for the penetration into the host and the survival of the saprophyte, but not for the infection and the expression of symptoms of verticillosis. It synthesizes ethylene, propyl alcohol, ethyl acetate, methyl acetate (phytotoxin).

The interaction between a plant and a microorganism therefore often results a change in the metabolism of one or both of the two organisms organisms at the same time. Furthermore, a symbiosis can result from the interaction between a plant and certain bacteria or certain fungi. Such natural processes are already advantageously exploited to improve the growth of certain plants. So, several examples of interaction between plants and bacteria have been described.

For example, rhizobacteria promote plant growth and protect them from pathogenic microorganisms. Pseudomonas fluorescens M.3.1 thus stimulates the growth of maize and limits the harmful effect of Fusadum roseum (Lugtenberg et al. 1991, Benizri et al. 1997).

Another effect of a bacteria promoting plant growth has been studied: Pseudomonas sp. PsJN strain increases the resistance of young tomato plants to Verticillium dahliae wilt (Sharma and Novak 1998). Tomato seedlings of a cultivar sensitive to Verticillium dahliae co-cultivated in vitro with the bacterium were infected with V. dahliae and seedlings colonized in vivo after 3 weeks of growth in the greenhouse. In in vitro culture , significant differences were noted between the plants co-cultivated with Pseudomonas and the control plants, the degree of protection conferred by bacterial colonization being a function of the density of the inoculum of V. dahliae . In in vivo culture , it is only after 3 weeks of exposure to the pathogen that growth differences appear. This suggests that endophytic colonization of tomato tissue by the bacteria is necessary for the expression of resistance to V. dahliae ( Sharma and Nowak 1998).

Other examples of in vitro "biotization" between plant tissues and microorganisms showing beneficial effects are described in a recent review article ( Nowak 1998 ): thus, a co-culture between soy cotyledons and two strains of Pseudomonas maltophilia stimulates the development of roots of Brassica campestris under gnotobiotic growth conditions.

The inventors have considered the possibility of exploiting the modifications metabolic resulting from the interaction between a plant and a microorganism, including a phytopathogen, to produce certain substances of interest, in a context favorable to exploitation industrial.

It is indeed impossible to envisage the cultivation in the open field of plants which would later be intentionally infected with phytopathogens. In effect, on the one hand, phytopathogens are organisms generally not very selective and would quickly infect other cultures than those targeted, and on the other hand, the agricultural zone would in fact be prohibited for the production of same plant genera.

The inventors therefore sought production conditions for substances of interest, compatible with a confined culture medium. The examples of mixed cultures associating two living partners in a confined volume, in order to collect the products of their metabolism are still very few and involve partners of the same biological kingdom.

William et al. describe for example co-cultures between bacteria making it possible to increase the use of xylan by Ruminococcus flavefaciens FDI. The culture of this bacterium in association with Methanobrevibacter smithii increases the specific activities of enzymes degrading extracellular polysaccharides. Interactions on hydrogen transfers occur, fermentation becomes acetogenic, methane is formed in place of hydrogen, allowing the accumulation of xylobiose and xylose. Similar effects are obtained by co-culture between Ruminococcus flavefaciens and Acetitomaculum ruminis (Williams et al. 1994) .

Mahagamasekera et al. describe an intergeneric coculture between roots transformed by Agrobacterium rhizogenes and Atropa belladonna and hybrid stems from Duboisia. This co-culture allows the production at a high level of scopolamine, while this substance is absent from cultures alone. In fact, plants producing scopolamine synthesize hyocyamine at the level of the roots, this precursor then being converted into scopolamine by an active enzyme mainly in the leaves. The co-culture of these two types of plant cells therefore restores the metabolic pathway, with synthesis of hyocyamine by the roots and conversion of hyocyamine to scopolamine by the stems (Mahagamasekera et al . 1998).

The present invention proposes to carry out co-cultures of tissues or cells of plant origin and tissues or cells of non-plant origin, such as as living phytopathogens, in fermenter, in order to produce substances of interest. The containment conditions allow bypass the "biosecurity" difficulties mentioned above. Co-culture in fermenter has other advantages, which will be presented further.

Examples of natural phytopathogens, which can be used in the context of the invention, are presented in Table 2 below, in relation to botanical families (hosts) known to be targeted by the latter.

These phytopathogenic / host pairs can be used in co-cultures of the invention.

However, the co-cultures of the present invention are not limited to co-cultures of cells of plant origin in the presence of a phytopathogen natural or proven from the plant in question. For example, a pathogen of a given botanical family can be co-cultivated with cells from from a different botanical family. We can also consider the co-culture of cells of plant origin with cells or organisms not corresponding not to a phytopathogen identified as such.

Indeed, although the only phytopathogenic plant cell interactions characterized to date are related to infections observable in the nature, the invention proposes to also produce cell co-cultures plants and cells with which they have never been contact, with the aim of inducing or promoting the synthesis of new compounds. This synthesis can under these conditions be the result of a de-repression of certain dormant metabolic pathways in one or more more than one cell type co-cultured or any other process metabolic.

In the remainder of this text, the term “phytopathogen” will therefore designate any organism or cells other than cells originating from higher plants, for example microorganisms such as bacteria, archabacteria, cyanobacteria, viruses, protists, yeasts, fungi, whether or not an organism capable of naturally infecting a plant, or cells isolated from multicellular organisms, in particular animal cells, as soon as the association in vitro, established under the conditions of the invention, between plant cells and the phytopathogen, leads to the production of substances of interest.

The term "authentic phytopathogen" will designate, within the framework of the definition given above of phytopathogens, a phytopathogen identified as such in the literal sense in the prior art, that is to say a organism, in particular microorganism or virus known to induce disease in a plant. In this definition, the term "disease" means any inhibition of growth, development, tissue necrosis, or decreased fertility in a plant. A phytopathogen will be said "Authentic" regardless of the extent of its natural host spectrum, from when this spectrum includes at least one plant species. A authentic phytopathogen can, according to the invention, be co-cultivated with plant cells of a species distinct from its natural host (s). Thus, Verticillium dahliae is an authentic phytopathogen since it is known to naturally parasitize a large number of plant species including dahlia, cotton, potato, cocoa, tomato, eggplant or the strawberry plant. In Example 2, this authentic phytopathogen is co-cultivated with Ruta graveolens cells, which is not its privileged natural host.

Where appropriate, the co-cultures of the invention may contain cells plants of several types and / or several different phytopathogens. In all cases, a co-culture according to the present invention will comprise at least one cell type of plant origin and one cell type of a different reign, the latter being designated by the term "Phytopathogenic".

The co-cultures of the present invention are "true" co-cultures, which means that the different cells and / or co-cultivated organisms are alive when brought to culture as opposed to elicitation by bringing plant cells into contact with inactivated natural extracts.

The co-cultures of the invention differ from cultures in which the living cells are either only plant cells or only phytopathogens, which can also be called "pure cultures". An example of a pure culture of phytopathogen is the culture of Verticillium dahliae described in Example 2. Another example of pure culture is that of a culture of plant cells in the presence of an elicitor such as autoclaved bacteria.

According to a particular embodiment of the invention, the co-culture is stable, which means that the different cell types present are capable of reproducing or at least subsisting in co-culture, so that a balance can be struck between the different lines (plants and phytopathogens) resulting in the fact that a large number of successive subcultures of the co-culture does not lead to the elimination of one of the lines. In practice, a co-culture can be considered stable when all the cell types present are alive after a complete cycle of plant cell growth authorizing the doubling of the reference cell population (when several types of plant cells are present in the coculture, the growth cycle considered will be that of the slowest cell). Indeed, experience shows that if all cell types of a co-culture containing plant cells and phytopathogens are living at the end of a slower cell type cell cycle, then the different cell types will be able to coexist and a balance will be struck between them. Conversely, if two cell types cannot coexist within of the same culture, this will result in rapid death (a few days at the maximum) of one of them.

In these co-cultures, cells of plant origin can be individualized or organized in multicellular structures. In particular, it can be dedifferentiated cells (example 2), or organs, by example of roots (example1).

By dedifferentiated plant cells is meant any plant cell that does not having none of the characteristics of a particular specialization and capable to live on its own and not in dependence on other cells. These dedifferentiated plant cells are possibly able, under the effect from an induction, to any differentiation consistent with their genome.

According to the culture method chosen, and in particular according to the environment of selected culture, it is possible to obtain from the same explant dedifferentiated plant cells with different characters (Plant propagation by tissue culture, George E. F. and Sherrigton P. D., 1984, Exegetics Limited).

In a preferred embodiment of the co-cultures of the invention, the phytopathogens are prokaryotic cells. In particular, it may be of bacteria naturally infecting the plants from which the cells come plants with which they are co-cultivated. However, co-cultures of the present invention are not limited to cell co-cultures of plant origin in the presence of a natural plant pathogen considered. For example, a pathogen from a given botanical family can be co-cultivated with cells from a different botanical family. It is also possible to envisage the co-culture of cells of plant origin with prokaryotic cells not corresponding to an authentic phytopathogen. A co-culture of plant cells and cells with which they do not have never been put in contact is indeed likely to modify certain dormant metabolic pathways in one or more of the cell types co-cultivated, and thus allow the synthesis of new compounds. of the co-cultures of cells of plant origin with infecting bacteria naturally from animals, or co-cultures of cells of plant origin with archabacteria, making it possible to produce substances of interest, are therefore also part of the present invention.

In another preferred embodiment of the co-cultures of the invention, the phytopathogens are fungi. In particular, it may be yeast or lower mushrooms.

As mentioned above, the co-cultures of the present invention are not limited to co-cultures of plant cells with a authentic phytopathogen. However, this type of co-cultures involving, on the one hand, plant cells (dedifferentiated or not, if necessary organized in multicellular structures) and, on the other hand, a phytopathogen authentic (whose natural plant host may or may not be distinct from cells particular plants of the co-culture), constitutes a particular aspect of the invention.

According to another particular embodiment of the invention, the co-culture is a stable co-culture of dedifferentiated cells and a phytopathogen any, in particular a phytopathogen which does not have a natural host, the particular plant cells of the coculture and, where appropriate, a non-authentic phytopathogen, i.e. not naturally infecting plants.

In another embodiment of the co-cultures of the invention, the phytopathogens are eukaryotic cells of animal origin. he can be primary cells or cells of a cell line immortalized. In addition, these cells can be genetically modified, possibly to secrete a compound that will interact with cells plants or their metabolites. Animal cells can be dedifferentiated or have characteristics of a particular cell type. These cells can for example be derived from mammals or insects. If necessary, they can be insects, in the larval state or not.

In a particular embodiment of the co-cultures of the invention, the phytopathogens are viruses. These viruses can be infecting viruses naturally plants, for example, among DNA viruses, those of the Caulimovirus family such as for example the mosaic virus cauliflower or, among RNA viruses, those of the family of Bromoviridae such as the alfalfa mosaic virus, without excluding viruses of a different natural tropism.

The number of cell types of plant origin present in co-cultures of the invention is not limited, nor is the number of types of phytopathogens co-cultivated with plant cells. For example, a co-culture of the invention may include a given type of plant cell, cells of an animal line, and an infecting virus naturally said cells of animal origin. Another example of complex culture according to the invention would include plant cells derived from two different botanical taxa, and a phytopathogen.

In a particular embodiment of the co-cultures defined above from the invention, the culture medium is advantageously initially perfectly defined. In particular, the culture medium used can be a fully synthetic medium. It can be chosen, for example, from culture media conventionally used to cultivate plant cells, but it can also be modified to allow optimized growth of different cell types co-cultivated.

Interestingly, the inventors have observed that phytopathogens can participate in the establishment of a stable co-culture with cells plants, in the presence of a culture medium which is not specific to them, even under cultivation conditions (immersion in the environment) special.

A particular co-culture of the invention is a culture of roots of Impatiens balsamina in the presence of Streptococcus sp. Another particular co-culture of the invention is a culture of Ruta graveolens dedifferentiated in the presence of Verticillium dahliae .

The co-cultures according to the invention are preferably produced in a fermenter which can be any type of enclosure. This enclosure is advantageously sterile and / or sterilizable, and / or agitated and / or subjected to agitation.

The substances of interest produced by the methods of the invention can be intended for several types of industrial applications, in particular in food, agrochemicals, pharmaceuticals and cosmetics. Preferred co-cultures of the invention are therefore co-cultures allowing the production of substances of interest for food, agrochemicals, pharmacy or cosmetics.

In a particular embodiment of co-culture according to the invention, a substance of interest produced by said co-culture is synthesized so more effective than in pure cultures in the context of the invention. The initiation or the increase of the synthesis of the substance of interest is measured against the pure culture that synthesizes said substance from the most effectively. The substance of interest can be released into the culture medium or not. In the latter case, a cell lysis step may be necessary to measure the production of the substance. In a preferred embodiment of co-culture according to the invention, the factor increase in the synthesis of a substance of interest is between 2 and 3, or between 3 and 10; in some cases it may be greater than 10 or even greater than 100.

A special type of substance that can be obtained from cocultures of the invention are the compounds with coloring power, usable in particular in cosmetic. Among these compounds, one can distinguish dyes and pigments. The dyes are soluble in a solvent and are molecules of reduced size which easily penetrate the hair. The pigments are insoluble in their environment of use. Their structure is crystalline or amorphous.

The first compounds with coloring power used were of vegetable origin (indigo, madder, logwood), animal (cochineal, purple), or mineral (overseas).

Table 3 below presents several compounds with coloring power, extracted from plants and fungi.

Currently, the majority of industrial dyestuffs are produced by chemical synthesis.

An application of the present invention is the obtaining of substances to coloring power from co-cultures involving plant cells.

This approach requires a priori consideration of the following:

Firstly, it is necessary to determine whether the co-cultures are feasible in the traditional media of one or other of the cell types cultured simultaneously or whether a consensual culture medium must be found, knowing that the media are very different for each of the cellular entities. Indeed, the two fields of microbiology and plant biotechnology have led to the development of specific culture media. Thus, many fungi are conventionally grown on potato carrot agar or potato dextrose agar (PDA) media, the preparation protocol of which is given below. These media are poorly controlled (complex media), due to the variability of the basic ingredients. Indeed, the potatoes and carrots used to prepare these environments may belong to different varieties and have been cultivated in multiple ways (soil, climate, soil improvers ...). On the other hand, plant cells can be cultured using well-defined media, for example of the Murashige & Skoog type, or Gamborg, for example. Conventionally, culture media for plant cells are perfectly defined media.

potato carrot agar : Pomme de terre : 20.0 g, Carottes : 20.0 g, Agar : 15.0 g, Eau distillée : 1.0 L

The composition of the culture media mentioned above is as follows:

potato carrot agar : Potato: 20.0 g, Carrots: 20.0 g, Agar: 15.0 g, Distilled water: 1.0 L

Cut the potatoes and carrots and cook them in water for 1/2 hour. Filter through a fine cloth and add the Agar.

potato dextrose agar (PDA)

Cut potatoes: 300.0 g, Glucose, 20.0 g, Distilled water 1.0L.

Boil the finely chopped potatoes in 500 ml of water until fully cooked. filter through a fine cloth and add water QSP 1.0 L. Heat to dissolve the Agar in the filtrate. Add glucose before sterilization.

Example of Gamborg base medium, (Murashige, Thorpe, Vasil, In Vitro, Vol. 12 (7): 473-478, 1976):

For 1 liter: Gamborg macro elements 100 ml Gamborg micro-element 1 ml Gamborg vitamins 2 ml EDTA iron 10 ml 2-4-dichloro phenoxy acetic acid 10 ^-4 (or 2,4-D10 ^-4 M) 10 ml Kinetin 10 ^-4 0.6 ml (0.06mg) Sucrose 20 g Distilled water qs 1 Liter pH before sterilization 5.8 UpH Sterilization 115 or 121 ° C for 20 to 40 minutes

To obtain an agar medium, Agar 8 g is added. Gamborg macro elements mg / l mM KNO ₃ 2500 25.0 CaCl ₂ , 2H ₂ O 150 1.0 MgSO ₄ , 7 H ₂ O 250 1.0 (NH ₄ ) ₂ SO ₄ 134 1.0 NaH ₂ PO ₄ , 1 H ₂ O 150 1.1 Gamborg microelements mg / l microM Kl 0.75 4.5 H ₃ BO ₃ 3.0 50.0 MnSO ₄ , 1 H ₂ O 10.0 60.0 ZnSO ₄ , 7H ₂ O 2.0 7.0 Na ₂ MoO ₄ , 2 H ₂ O 0.25 1.0 CuSO ₄ , 5 H ₂ O 0025 0.1 CoCl ₂ , 6 H ₂ O 0025 0.1 Gamborg vitamins mg / l microM MYO - INOSITOL 100.0 555.5 NICOTINIC ACID 1.0 8.0 PYRIDOXIN HCI (B ₆ ) 1.0 4.8

Example of Murashige & Skoog Base midfielder (Detailed)

Skoog macro elements 100 ml / l Skoog micro elements 1 ml Vitamins of Skoog 2 ml EDTA iron 10 ml 2-4 D 10 ^-4 10 ml Kinetin 10 ^-4 0.6 ml Sucrose 30 g Distilled water qs 1 litre pH before sterilization 5.8 UpH Sterilization 115 or 121 ° C for 20 to 40 minutes

To obtain a solid agar medium, add: agar 8 g Macro elements in mg / l Skoog KNO ₃ 1900 NH ₄ NO ₃ 1650 MgSO ₄ , 7 H ₂ O 370 CaCl ₂ , 2 H ₂ O 440 KH ₂ PO ₄ 170 Micro elements in mg / l Skoog CuSO ₄ , 5 H ₂ O 0025 MnSO ₄ , 1 H ₂ O 16.9 Kl 0.83 Na ₂ MoO ₄ , 2 H ₂ O 0.25 ZnSO ₄ , 7 H ₂ O 10.6 H ₃ BO ₃ 6.2 CoCl ₂ , 6 H ₂ O 0025 Vitamins mg / l Skoog myo 100 Nicotinic acid 0.5 pyridoxine 0.5 thiamin 0.1 FeSO ₄ , 7 H ₂ O 27.8 Na ₂ EDTA 37.3

Besides the question of the co-culture environment, it is necessary to determine whether it is possible to establish stable co-cultures , in the sense that a balance exists between the different lines and that none of them is eliminated. during successive subcultures of the coculture.

Finally, the production of the metabolites sought may, according to the associations performed, be uncertain and should be verified.

The surprising results obtained and presented in the detailed examples below, allow these three questions to be answered. These examples show in particular that it is possible, contrary to all expectations, to co-cultivate stably plant cells and bacteria, in a classical medium for the cultivation of plant cells, and that this co-culture allows the production of substances that are not produced efficiently in pure culture of said plant cells and bacteria (Example 1). Example 2 shows, again contrary to all expectations, that co-culture plant cells and fungi in a classic culture medium of plant cells, can allow the synthesis of compounds absent in pure cultures of said plant and fungi cells.

The present invention therefore relates to methods of producing substances of interest, by co-culture of plant cells and live plant pathogens, as defined and under the conditions described above for co-cultures. The methods of the invention can in particular allow the production of substances of pharmaceutical interest and / or cosmetic, in particular the production of substances with power dye.

In a preferred embodiment of the methods of the invention, the co-culture of plant cells and living plant pathogens is stable, which means that a balance is struck between the different lines, which remain present and alive during the subculture subculture. As stated more high, this stability is ensured when all the cell types of the co-cultures are alive after a complete cycle of the most cell type slow.

In a preferred implementation of the methods of the invention, the co-culture is carried out in a sterile and / or sterilizable fermenter or sealed enclosure, shaken and / or shaken. Examples of usable fermenters in the processes of the invention are brand stirring fermenters RUSHTON, AIRLIFT, DRAFT Tube (DRAUGHT Tube in the USA) or even piston type. In the methods of the invention, the organisms can be grown together or separated by a membrane in a batch system, fed batch or continuous. Physical processes to separate two cultures by a membrane are described in US Patent 5,665,596.

In a batch system, the cells multiply in the fermenter up to exhaustion of the culture medium.

In a fed batch system, at the end of batch culture, part of the culture is collected (from 50 to 80% in general). The volume withdrawn is replaced by new middle. Several identical cycles are possible.

In a continuous system, at the end of the exponential phase of culture in mode batch, the fermenter is continuously harvested and at the same time filled with same flow in new medium as the flow taken.

These 3 production techniques must be carried out axenically.

In a preferred implementation of the methods of the invention, the co-culture is carried out in an initially perfectly defined culture medium. In particular, the culture medium used can be an entirely synthetic. It can be chosen for example from culture media conventionally used to cultivate plant cells. Of course, the co-culture leads to the production of substances which will subsequently this more complex environment. The molecules produced by each of the types cell cultures can therefore have an effect, direct or indirect, on cells of the other type.

In the methods of the invention, plant cells can be isolated or organized in multicellular structures. In particular, it may be dedifferentiated cells (example 2), or organs, for example roots (Example1).

In describing the methods of the present invention, the term "Phytopathogenic" is not limited to naturally capable organisms to infect plants (authentic phytopathogens), even if the processes involving authentic phytopathogens constitute a particular class of processes according to the invention. The invention also relates to processes for the production of substances of interest by co-cultivation of plant cells with cells with which they have never been contact. In the methods of the invention, cells of plant origin are co-cultivated with cells, tissues or organisms which can be chosen among archabacteria, cyanobacteria, and in general, bacteria, yeast, fungi, animal cells, insects or viruses. These cells, tissues or organisms are designated here by the term "Phytopathogenic".

In a particularly preferred implementation of the methods of the invention, the co-culture of plant cells and phytopathogens allows synthesis of substances which are not produced in pure culture any of the co-cultivated elements, or which are produced at low levels. Co-culture then allows a living-living interaction between these elements, which advantageously induces a modification of the metabolism one or more of the co-cultured cell types, resulting in increased synthesis of interesting substances. When putting implementing the methods of the invention, the synthesis of a substance interesting can be increased by a factor 2 to 3, or by a factor 3 to 10, or even by a factor greater than 10 and, where applicable, by a factor greater than 100.

Substances of interest whose synthesis is favored by a co-culture according to the methods of the invention can be part of the group including phytoalexins, quinones and their derivatives, lawsone, polyphenol oxidases, furocoumarins, phytochelatins, peptides and / or proteins.

A. Ensemencement du milieu de culture avec les cellules végétales et les phytopathogènes choisis,

B. Co-culture des cellules végétales et des phytopathogènes, pendant 1 à 30 jours, en batch et durant toute la production en mode continu,

C. Récupération de la substance recherchée à partir du milieu de culture.

A method of the invention comprises for example the following steps:

A. Inoculation of the culture medium with the plant cells and the phytopathogens chosen,

B. Co-culture of plant cells and phytopathogens, for 1 to 30 days, in batch and during all production in continuous mode,

C. Recovery of the desired substance from the culture medium.

In a particular implementation of the methods of the invention, a coloring substance is produced by co-cultivation of roots of Impatiens balsamina and Streptococcus sp (example 1). In another particular implementation of the methods of the invention, cells of Ruta graveolens are cultured in the presence of Verticillium dahliae , which leads to an increase in the production of flavioline by Verticillium dahliae , and an increase in the production of furocoumarins by Ruta graveolens.

Of course, the substances of interest obtained by any of the The methods of the invention also form part of the present invention. this is especially true for phytoalexins and coloring substances obtained by these methods.

The examples and figures below illustrate the implementation and the advantage of the present invention, without however limiting its scope.

Figure 1A : Chromatogramme d'une solution de lawsone pure

Figure 1B : Chromatogramme du milieu de culture vierge

Figure 1C: Chromatogramme de racines d'Impatiens balsamina autoclavées, mises en présence d'un Streptocoque vivant

Figure 1D : Chromatogramme de racines d'Impatiens balsamina élicitées par le Streptocoque autoclavé

Figure 1E : Chromatogramme de racines d'Impatiens balsamina saines, en culture seule

Figure 1F : Chromatogramme d'une co-culture de racines d'Impatiens balsamina contaminées par le Streptocoque vivant

Figure 1 G: Chromatogramme de racines d'Impatiens balsamina recontaminées par le Streptocoque vivant

The chromatograms presented in FIG. 1 (ethanolic extraction) show the influence of the different culture conditions on the composition of the roots of Impatiens balsamina.

Figure 1A: Chromatogram of a pure lawsone solution

Figure 1B: Chromatogram of virgin culture medium

Figure 1C: Chromatogram of autoclaved Impatiens balsamina roots, placed in the presence of a living Streptococcus

Figure 1D: Chromatogram of Impatiens balsamina roots elicited by autoclaved Streptococcus

Figure 1E: Chromatogram of roots of healthy Impatiens balsamina , grown alone

Figure 1F: Chromatogram of a co-culture of roots of Impatiens balsamina contaminated with live Streptococcus

Figure 1 G: Chromatogram of roots of Impatiens balsamina recontaminated by living Streptococcus

EXAMPLES Example 1 In vitro co-cultures of roots of Impatiens balsamina and Streptococcus sp . :

Impatiens balsamina belongs to the Balsaminaceae family. This plant produces among other things a coloring substance found in Henna: lawsone or 2 hydroxy 1,4 naphytoquinone. Root strains obtained in the laboratory also produce lawsone in small quantities.

Surprisingly, the inventors observed two different behaviors of the same root strain of Impatiens balsamina cultivated in the same way in 250 ml Erlemeyer on a rotary shaker transplanted every 14 days on the same basic medium which is as follows: CaCl ₂ , 2H ₂ O 0.44 g / L KH ₂ PO ₄ 0.17 g / L MgSO ₄ , 7H ₂ O 0.37 g / L NH ₄ NO ₃ 1.65 g / L CuSO ₄ , 5H ₂ O 0025 mg / L CoCl ₂ , 6H ₂ O 0025 mg / L Na ₂ MoO ₄ , 2H ₂ O 0.25 mg / L Kl 0.83 mg / L ZnSO ₄ , 7H ₂ O 8.6 mg / L H ₃ BO ₃ 6.2 mg / L MnSO ₄ , 4H ₂ O 22.3 mg / L myo 100 mg / L Nicotinic acid 0.5 mg / L Pyridoxine, HCI (4 ° C) 0.5 mg / L Thiamine, HCI (4 ° C) 0.1 mg / L wistaria 2 mg / L FeSO ₄ , 7H ₂ O 27.8 mg / L Sequestrene 330 Fe 37.3 mg / L Naphthalene Acetic Acid 1 mg / L kinetin 0.06 mg / L Sucrose 30 g / L Distilled water qsp 1 Liter pH before sterilization 5.8 UpH Sterilization according to volumes: from 15 'to 40' at 115 ° C or 121 ° C

The roots are inoculated at the rate of 5 g of fresh material per 100 ml of culture.

One of the two cultures has beige roots while the second is orange red. The two cultures have been stable for several years.

The red culture was filtered and its medium was analyzed. The latter contains bacteria. Isolation could reveal that it is a pure bacterial culture which has been identified by the Pasteur Institute as being a Streptococcus sp.

The latter was grown in the dark at 26.5 ° C on LPG medium, more conducive to its development, of the following composition: Yeast extract 5 g / L Glucose 10 g / L Peptone 5 g / L agar 15 g / L Culture on agar Distilled water qsp 1 Liter Sterilization according to volumes: from 15 'to 40' at 115 ° C or 121 ° C.

To verify that the presence of the red coloration of the Impatiens balsamina strain is actually induced by the bacteria, beige roots (uncontaminated) were infected with Streptococcus cultivated on LPG medium. In all cases of infection, the appearance of the red color is observed.

The phenomenon is stable over the subcultures, and a true and stable co-culture sets up, preserving the roots of Impatiens balsamina their red coloration.

These same bacteria, killed by heat and then added to non-pigmented roots, produce no change. The table below shows the different tests carried out:

The phenomenon is therefore not due to a traditional elicitation but rather to a true co-culture of living cells.

The first surprising effect observed here is the adaptation of Streptococci to plant cell culture medium.

The second surprising effect is the stability of co-cultures over time: neither of the two cell lines takes precedence over the other.

Different dosages of Lawsone (yellow / orange) show that it can be elicited by the killed streptococcus, but this cannot explain the appearance bright red coloration of the roots obtained in co-culture.

The chromatograms presented in FIG. 1 (ethanolic extraction) show the influence of the different culture conditions on the composition of the roots of Impatiens balsamina .

EXAMPLE 2 In Vitro Co-Cultures of Ruta graveolens (Rhue) and Verticillium Dahliae Cells

This example illustrates the possibility of cultivating a dedifferentiated cell of plant origin in the presence of a phytopathogenic fungus.

Ruta graveolens is a plant from the Rutaceae family which synthesizes furocoumarins including psoralen and some of its methoxylated derivatives: 5-MOP (bergaptene), 8-MOP (xanthotoxine) and 5,8-MOP (isopimpinellin). These furocoumarins are secondary metabolites produced in response to an attack by a phytopathogen. They are therefore phytoalexins limiting the proliferation of phytopathogens.

Verticillium dahliae is a phytopathogenic lower fungus belonging to the family of Adelomycetes of the order of the Hyphales parasitizing a large number of plant species including dahlia, cotton, potato, cocoa, tomato, eggplant or even Strawberry plant. Among other things, it synthesizes a naphthoquinone: flaviolin or 2,5,7 trihydroxy 1,4 naphtoquinone.

Its culture is carried out on a PDA medium of composition: Mashed potatoes 200 g / L Glucose 20 g / L (Agar 15 g / L) pH before sterilization 5.6 UpH

Cultures are carried out in the dark in a Petri dish at 26.5 ° C.
Its passage in liquid medium takes place in medium for plant growth B5 D2 in the dark:

Macro-, micro-elements, vitamins and iron from the middle of Gamborg, 2,4-dichlorophenoxyacetic acid 10 ^-4 M 2 mg / L Sucrose 30 g / L PH before sterilization 5.8 UpH

Ruta graveolens is also grown on B5 D2 medium in the light.

Pure cultures of each of the cell entities are carried out, cultures elicited by cells of the other type killed by heat and finally the true co-culture between Ruta graveolens and Verticillium dahliae.

Flavioline : extracellulaire

Furocoumarines : intracellulaires

MS : Matière sèche en cellule végétale

Each cell entity is extracted and then analyzed. The results are as follows:

Flavioline: extracellular

Furocoumarins: intracellular

MS: Dry matter in plant cells

The tests were carried out in an illuminated culture more suited to the growth of the strain of R. graveolens

These experiments show that the production of flavioline is optimized in true co-culture conditions. In addition, frozen cells (not viable) give the same results as pure mushroom cultivation. This result clearly shows that living / living interaction is necessary for increase synthesis by a factor of 2.5. Autoclaving shows a denaturation of the components of the plant cell. These last ones behave as less effective elicitors than true co-culture.

With regard to the production of furocoumarins, these results show that the addition of killed fungus to the culture of Ruta graveolens has the opposite effect to that sought, since only a third of the furocoumarins produced in pure culture are produced. Co-culture makes it possible to produce 3 times more furocoumarins than pure culture.

These results therefore clearly show the effects of the interaction. living / living, which allows an increase in the biosynthesis of compounds in the two cell lines. These compounds are very different: dyes and phytoalexins, thus showing the richness of this type of technique.

Claims (31)

Stable co-culture in vitro of cells of plant origin and phytopathogens, allowing the production of substances of interest. Co-culture according to claim 1, characterized in that the cells of plant origin are individualized or organized into multicellular structures. Co-culture according to claim 1 or 2, characterized in that the plant cells are dedifferentiated. Co-culture according to claims 1 to 3, characterized in that the phytopathogens are archabacteria, bacteria, protists, fungi, animal cells, insects, viruses or yeasts. Co-culture according to claims 1 to 4, in which the phytopathogens are prokaryotic cells. Co-culture according to claims 1 to 4, in which the phytopathogens are fungi. Co-culture according to claims 1 to 4, in which the phytopathogens are viruses. Co-culture according to any one of claims 1 to 7, characterized in that the phytopathogens are authentic phytopathogens. Co-culture according to claims 1 to 4, in which the phytopathogens are yeasts. Co-culture according to claims 1 to 5, in which the plant cells are roots of Impatiens balsamina and the phytopathogen is Streptococcus sp. Co-culture according to claims 1 to 4 and 6, in which the plant cells are dedifferentiated Ruta graveolens cells and the phytopathogen is Verticillium dahliae. Co-culture according to any one of claims 1 to 11, characterized in that the culture medium is initially perfectly defined. Co-culture according to any one of Claims 1 to 12, allowing the production of substances of interest for food, agrochemicals, pharmacy or cosmetics. Co-culture according to any one of Claims 1 to 13, characterized in that it produces a substance of interest more effectively than the pure cultures of said plant cells or of said phytopathogens of said co-culture, the factor of increase in the synthesis of said substance of interest by said co-culture, compared to the most effective pure culture, being between 2 and 3, or between 3 and 10, or greater than 10, or greater than 100. Process for producing fermenters, substances of interest, by co-culture living plant cells and phytopathogens. Use of a process according to claim 15 for the production substances of interest for food, agrochemistry, pharmacy and / or cosmetics. Use of a process according to claim 15 for the production of substance with coloring power. Method or use according to claims 15 to 17, wherein the co-culture is stable. Method or use according to claims 15 to 18, wherein the co-culture is carried out in a sterile fermenter or sealed enclosure and / or sterilizable, agitated and / or subjected to agitation. Method or use according to claims 15 to 19, wherein organisms are grown together or separated by a membrane in batch, fed batch or continuous system. Method or use according to claims 15 to 20, wherein the culture medium is initially perfectly defined. Method or use according to claims 15 to 21, wherein plant cells are isolated or organized into structures multicellular. Method or use according to claims 15 to 21, wherein plant cells are dedifferentiated. Method or use according to claims 15 to 23, wherein phytopathogens are chosen from archabacteria, bacteria, protists, fungi, animal cells, insects or viruses. Method or use according to claims 15 to 24, wherein phytopathogens are authentic phytopathogens. Method or use according to claims 15 to 25, comprising the following steps: A. Inoculation of the culture medium with the plant cells and the phytopathogens chosen, B. Co-culture of plant cells and phytopathogens, for 1 to 30 days in batch, and during all production in continuous mode, C. Recovery of the desired substance from the culture medium. Process or use according to Claims 15 to 26, for the production of a coloring substance by co-cultivation of roots of Impatiens balsamina and Streptococcus sp. The method of claims 15 or 18 to 27, wherein the co-culture of plant cells and phytopathogens allows produce a substance of interest more efficiently than pure cultures of said plant cells or phytopathogens of said co-culture, the factor for increasing the synthesis of said substance of interest by said co-culture, compared to pure culture the most effective, being between 2 and 3, or between 3 and 10, or greater than 10, or greater than 100. Use of a method according to claims 15 or 18 to 28 in which the co-culture of plant cells and phytopathogens allows the synthesis of substances which are not produced effectively in a pure culture. Use according to claim 28, characterized in that said substances are part of the group comprising phytoalexins, quinones and their derivatives, lawsone, polyphenol oxidases, furocoumarins, phytochelatins, peptides or proteins Phytoalexins and coloring substances obtained by any one from methods 15 or 18 to 27.

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